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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v
Toxicity of a glufosinate- and several glyphosate-based
herbicides to juvenile amphibians from the Southern High
Plains, USA
Simon K. Dinehart a,⁎, Loren M. Smith a , Scott T. McMurry a , Todd A. Anderson b ,
Philip N. Smith b , David A. Haukos c
a
Postal address: Department of Zoology, Oklahoma State University, Stillwater, OK 74078, USA
The Institute of Environmental and Human Health, Texas Tech University, Box 41163, Lubbock, TX 79409, USA
c
U.S. Fish and Wildlife Service, MS 2125, Texas Tech University, Lubbock, TX 79409, USA
b
AR TIC LE D ATA
ABSTR ACT
Article history:
Pesticide toxicity is often proposed as a contributing factor to the world-wide decline of
Received 1 August 2008
amphibian populations. We assessed acute toxicity (48 h) of a glufosinate-based herbicide
Received in revised form
(Ignite® 280 SL) and several glyphosate-based herbicide formulations (Roundup
26 September 2008
WeatherMAX®, Roundup Weed and Grass Killer Super Concentrate®, Roundup Weed and
Accepted 1 October 2008
Grass Killer Ready-To-Use Plus®) on two species of amphibians housed on soil or moist
paper towels. Survival of juvenile Great Plains toads (Bufo cognatus) and New Mexico
spadefoots (Spea multiplicata) was reduced by exposure to Roundup Weed and Grass Killer
Keywords:
Ready-To-Use Plus® on both substrates. Great Plains toad survival was also reduced by
Amphibian
exposure to Roundup Weed and Grass Killer Super Concentrate® on paper towels. New
Glufosinate
Mexico spadefoot and Great Plains toad survival was not affected by exposure to the two
Glyphosate
agricultural herbicides (Roundup WeatherMAX® and Ignite® 280 SL) on either substrate,
Ignite
suggesting that these herbicides likely do not pose an immediate risk to these species under
Roundup
field conditions.
© 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Amphibian populations are declining worldwide (Wyman,
1990), due in large part to the degradation of wetland and
terrestrial habitats (e.g.,Wyman, 1990). Chemicals, such as
insecticides, herbicides, and fertilizers used in agricultural
activities may also contaminate aquatic and terrestrial
habitats required by amphibians and pose a threat via direct
toxicity (Semlitsch, 2003). Glyphosate (e.g., Roundup®) and
glufosinate-ammonia (e.g., Ignite®) based herbicides are used
worldwide (Howe et al., 2004; Lee et al., 2005) to control weeds
in farmland and forests (Lee et al., 2005; Relyea 2005a).
Glyphosate-based herbicides are also frequently applied in
residential settings (Relyea, 2005a).
Most glyphosate-based herbicides contain two basic
components: the isopropylamine (IPA) salt of glyphosate
and a surfactant (the most common being a polyethoxylated
tallowamine, POEA, surfactant) (Giesy et al., 2000). Glufosinate herbicides contain glufosinate-ammonium and a
sodium polyoxyethylene alkylether sulfate (AES) surfactant
(Koyama and Goto, 1997). Both glyphosate and glufosinateammonium adsorb strongly to soil (Malone et al., 2004; Lee et
al., 2005), degrade rapidly via microbial activity and have
limited environmental persistence (Faber et al., 1997; Giesy et
⁎ Corresponding author. Tel.: +1 405 744 5555; fax: +1 405 744 7824.
E-mail address: simon.dinehart@okstate.edu (S.K. Dinehart).
0048-9697/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2008.10.010
Please cite this article as: Dinehart SK, et al, Toxicity of a glufosinate- and several glyphosate-based herbicides to juvenile
amphibians from the Southern High Plains, USA, Sci Total Environ (2008), doi:10.1016/j.scitotenv.2008.10.010
ARTICLE IN PRESS
2
SC IE N CE O F THE TOTA L E N VI RON ME N T XX ( 2 00 8 ) XXX–X XX
al., 2000). In terrestrial situations, the POEA surfactant
displays environmental fate similar to glyphosate (Giesy et
al., 2000). Little information on the fate of the surfactant used
in glufosinate herbicides is available. Since the major
components of glyphosate herbicides bind tightly to soil
and rapidly degrade, it is often assumed that they pose little
risk to non-target organisms (Relyea, 2005a). However, recent
work indicates that exposure to these chemicals can negatively affect amphibians within terrestrial (Relyea, 2005a) and
aquatic habitats (Howe et al., 2004; Relyea, 2004; 2005a).
Numerous studies have investigated effects of glyphosate
formulations on larval amphibians and results indicate that
the surfactants, rather than the active ingredient, may be
responsible for observed mortalities (Mann and Bidwell, 1999;
Howe et al., 2004; Relyea, 2004; Relyea et al., 2005; Relyea,
2005a,b). Non-ionic surfactants, such as POEA, exhibit their
negative effects primarily by disrupting the respiratory
surfaces of aquatic organisms (Lindgren et al., 1996). Following
metamorphosis, many amphibian species occupy terrestrial
habitats. Yet few studies (Bidwell and Gorrie, 1995; Mann and
Bidwell, 1999; Relyea, 2005a) have examined how postmetamorphic amphibians are affected by exposure to commonly applied herbicides. No work has examined whether
natural environmental factors (e.g., soil) modulate the toxicity
of herbicides toward post-metamorphic amphibians. Further
research conducted under increasingly realistic conditions is
necessary to fully understand how common agrochemicals
affect amphibians (Relyea, 2005a).
Our purpose was to estimate juvenile survival of two of the
most abundant amphibian species (Spea multiplicata, New
Mexico spadefoot; Bufo cognatus, Great Plains toad) from
playa wetlands of the Southern High Plains (SHP) following
exposure to common herbicides at environmentally relevant
levels. The SHP of Texas and New Mexico is one of the most
heavily cultivated regions in the world (Bolen et al., 1989). It is
therefore not surprising that the total volume of pesticides
applied in Texas is among the greatest in the United States
(Gianessi and Marcelli, 2000). Application to cotton represents
one of the most prevalent uses of glyphosate-based herbicides
(National Pesticide Use Database, 2004).
Because the nearly 25,000 SHP playas are principally
embedded throughout an intensively farmed region, terrestrial margins of many playas likely receive overspray during
applications of agrochemicals. Following metamorphosis,
juvenile amphibians inhabit areas near playas while the soil
remains moist (Voss, 1961; Graves and Kruppa, 2005; Morey,
2005). New Mexico spadefoots and Great Plains toads often
occupy shallow burrows (Degenhardt et al., 1996) and emerge
primarily for nocturnal foraging (Bragg, 1944; Garrett and
Barker, 1987). However, recently metamorphosed individuals
may also disperse away from drying playas (Degenhardt et al.,
1996). Due to this behavior and the fact that herbicides are
applied to cotton at various times throughout the spring and
summer (National Research Council, 1975; Bayer CropScience
LP, 2005; Monsanto Company, 2005), juvenile SHP amphibians
may be exposed to common herbicides. During our study,
juvenile amphibians were exposed to environmentally relevant concentrations of a glufosinate-ammonium based herbicide [Ignite® 280 SL (IG)] and several glyphosate-based
herbicide formulations [Roundup WeatherMAX® (WM),
Roundup Weed and Grass Killer Super Concentrate ®
(WGKC), and Roundup Weed and Grass Killer Ready-To-Use
Plus® (WGKP)] while housed on moist paper towels or natural
soil and survival was monitored for 48 h following
application.
2.
Materials and methods
Recently metamorphosed Plains and New Mexico spadefoot
toads were collected on 27 June 2007 adjacent to a cropland
playa wetland in Hale County, TX, USA. A mixture of the two
species was collected because at a young age the two are
difficult to distinguish (Degenhardt et al., 1996). Great Plains
toad juveniles were collected near a cropland playa in Hale
County, TX on 8 July 2007. Similar sized individuals were
collected to ensure that animals used for subsequent toxicity
testing were of similar developmental stage. The specific
exposure history of the populations from which animals used
in this study were drawn is unknown. However, these
amphibian populations likely experienced previous pesticide
exposure because they inhabit wetlands surrounded by
agriculture. All subsequent animal care and experimental
procedures (with exceptions noted) were the same for both
spadefoot and Great Plains toads. This research was completed under a Texas Tech University Institutional Animal
Care and Use Committee approved protocol (No. 06018-06).
After collection, animals were transported to The Institute of
Environmental and Human Health at Texas Tech University in
Lubbock, TX. They were held in 37.9 L glass aquaria containing
6 cm of moistened natural soil obtained from Terry County,
TX. The physiochemical characteristics of this sandy loam soil
were previously determined by A&L Midwest Laboratories
(Omaha, NE). The soil displayed the following properties: 74%
sand, 10% silt, and 16% clay, 1.3% organic matter, and pH of 8.3
(Zhang et al., 2006). Though this soil was not tested for
glyphosate- or glufosinate-based herbicide residues, significant chemical contamination is unlikely because the soil was
obtained from an area where no pesticides have been applied
for at least five years. Small crickets were provided ad libitum
to juveniles throughout the following experiments. Fluker's
Orange Cube Complete Diet (Fluker's Cricket Farm, Inc., Port
Allen, LA) was provided to all crickets for at least 6 h.
Spadefoot and Great Plains toads were allowed to
acclimate to laboratory conditions for three and four days,
respectively. The spadefoot toad experiment commenced on
30 June 2007, while that with Great Plains toads began 13 July
2007. Experimental compartments were 11.4 L (31.5 cm long
by 20.1 cm wide) plastic tubs lined with either paper towel or
the previously described natural soil (260 g – dry weight). The
soil covered the bottom of each tub evenly without allowing
metamorphs to bury themselves. A 946.4 mL (32 oz) garden
spray bottle was used to spray both substrates with aged well
water until they were visibly moist. Paper towel lined
containers received 14 g of evenly dispersed water, while
soil lined containers received 28 g of water. Ten randomly
selected juveniles were then added to each tub and allowed
to acclimate for 6 h prior to herbicide application. Due to a
counting error, a single tub received only nine spadefoot
juveniles.
Please cite this article as: Dinehart SK, et al, Toxicity of a glufosinate- and several glyphosate-based herbicides to juvenile
amphibians from the Southern High Plains, USA, Sci Total Environ (2008), doi:10.1016/j.scitotenv.2008.10.010
ARTICLE IN PRESS
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Table 1 – List of ingredients present (by percent
composition) in each herbicide formulation sprayed onto
juvenile Spea multiplicata (New Mexico spadefoot) and
Bufo Cognatus (Great Plains toad)
Herbicide
formulationa
WM
WGKP
WGKC
IG
a
Ingredient
Percent
Glyphosate
Other ingredients
Glyphosate
Pelargonic and related fatty acids
Water and minor formulating
ingredients
Glyphosate
Other ingredients
Glufosinate-ammonium
Other ingredients
48.8
52.2
2
2
96
50.2
49.8
24.5
75.5
®
WM = Roundup WeatherMAX , WGKP = Roundup Weed and Grass
Killer Ready-To-Use Plus®, WGKC = Roundup Weed and Grass Killer
Super Concentrate®, IG = Ignite® 280 SL.
All herbicides were applied at the maximum rate allowed
for a single application. This was done to simulate direct
exposure by terrestrial overspray (Relyea 2005a; Table 1); our
study therefore represented a “worst-case” exposure scenario.
WM was applied at a rate of 0.16 mL glyphosate/m2 (44 fl oz
WM/ac) (Monsanto Company, 2005), WGKC at a rate of 1.33 mL
glyphosate/m2 (2.5 fl oz WGKC/ft2) (Monsanto Company, 2006),
and IG at 0.21 mL glufosinate/m2 (29 fl oz IG/ac) (Bayer
CropScience LP, 2005). Because no application rate was
provided for WGKP, it was applied at a rate (based on amount
of glyphosate) equivalent to that recommended for the other
residential-use formulation (WGKC).
Herbicide solutions were applied using 946.4 mL garden
spray bottles. Initially, each bottle was calibrated so that 10
sprays delivered a consistent amount of water into an empty
11.4 L tub. Over the course of six such 10-spray trials, the
amount of water delivered by each bottle was consistent
among treatments (mean ± 1 standard error: WM, 8.64 ± 0.03 g;
WGKC, 8.73 ± 0.02 g; WGKP, 8.32 ± 0.02 g; IG, 8.96 ± 0.04 g; aged
well water, 8.74 ± 0.01 g). Herbicides were diluted with aged
well water so that they could be applied at the previously
stated rate. The proper dilution for each herbicide was
determined as follows. Based on the desired application rate
for each product, the amount of herbicide required for an area
the size of our experimental tubs (633.15 cm2) was calculated.
Pure herbicide was then diluted so that 10 sprays from the
appropriate bottle would deliver the desired amount of
herbicide to each tub. Spray bottles were filled with diluted
herbicide solution, the calibration of each was checked, and
adjustments were made if necessary. Herbicides were then
applied to experimental tubs via 10 evenly spaced sprays from
the appropriate bottle. Control tubs received 10 sprays of well
water. All calibration and herbicide applications were performed by the same person. There were five treatments (four
herbicide formulations plus a control) that were replicated
four times for each of two substrates (soil or paper towel) for
each species.
Following herbicide application, survival was monitored
for 48 h to assess the acute response to herbicide exposure.
Tubs were checked every 6 h and moribund individuals
Fig. 1 – The survival (mean ± 1 standard error) of juvenile Spea
multiplicata (New Mexico spadefoot) 48-h after direct exposure
to aged well water (control) or an herbicide at the given rate:
Roundup Weed and Grass Killer Ready-To-Use Plus® (WGKP),
1.33 mL glyphosate/m2; Roundup Weed and Grass Killer
Super Concentrate® (WGKC), 1.33 mL glyphosate/m2;
Roundup WeatherMAX® (WM), 0.16 mL glyphosate/m2; Ignite®
280 SL (IG), 0.21 mL glufosinate/m2. Animals were exposed
in plastic tubs lined with soil (shaded bars) or paper towel
(open bars).
euthanized by immersion in a 1% MS-222 solution (Howe
et al., 2004). Animals were considered moribund if they
exhibited lethargy or non-responsiveness to prodding. At the
end of the experiment, all remaining spadefoot toads were
euthanized. Protein electrophoresis, following the techniques
of Simovich and Sassaman (1986), was used to identify
juveniles as New Mexico or Plains spadefoots. All spadefoot
juveniles were weighed at the time of death. The mean mass
of Plains spadefoots (± 1 standard error) was 1.81 ± 0.04 g, and
that of New Mexico spadefoots was 1.85 ± 0.02 g. Great Plains
toads were weighed as they were distributed to experimental
tubs; mean mass (±1 standard error) was 0.74 ± 0.01 g.
Electrophoresis identified 337 of the spadefoot juveniles as
New Mexico spadefoots, 59 as Plains spadefoots, and 2 as
hybrids. Because New Mexico spadefoots dominated all
Table 2 – Pair-wise comparisons of mean survival of
juvenile Spea multiplicata (New Mexico spadefoot) 48-h
after direct exposure to aged well water (control) or an
herbicide a in plastic tubs
Contrast b
Df
χ2
P
Control vs. IG
Control vs WM
Control vs WGKC
Control vs WGKP
1
1
1
1
0.01
0.19
0.81
79.49
0.91
0.66
0.37
b0.0001
The degrees of freedom (df), test statistic (χ2) and associated
probability (P) are given.
a
Ignite® 280 SL (IG), Roundup WeatherMAX® (WM), and Roundup
Weed and Grass Killer Super Concentrate® (WGKC) were applied at the
maximum rate allowed for a single application: IG = 0.21 mL
glufosinate/m2, WM = 0.16 mL glyphosate/m2, WGKC = 1.33 mL
glyphosate/m2. Roundup Weed and Grass Killer Ready-To-Use Plus®
(WGKP) was applied at a rate equivalent to that recommended for
WGKC.
b
Means were compared with a CONTRAST statement in GENMOD.
Please cite this article as: Dinehart SK, et al, Toxicity of a glufosinate- and several glyphosate-based herbicides to juvenile
amphibians from the Southern High Plains, USA, Sci Total Environ (2008), doi:10.1016/j.scitotenv.2008.10.010
ARTICLE IN PRESS
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SC IE N CE O F THE TOTA L E N VI RON ME N T XX ( 2 00 8 ) XXX–X XX
experimental tubs, statistical analysis was only possible for
this species and Great Plains toads. Generalized linear model
(PROC GENMOD, SAS Version 9.1, SAS Institute, Cary, NC),
assuming a poisson distribution with a log link function (Littell
et al., 2002), were used to test whether New Mexico spadefoot
and Great Plains toad survival was influenced by pesticide
exposure. Number of surviving juveniles was the response
variable, and herbicide formulation and substrate (soil or
paper towel) were the treatment effects. Since the data
contained many zeros, 0.001 was added to each data value
so that the GENMOD model converged. Treatment means
(number surviving) were separated by including CONTRAST
statements in the GENMOD procedure.
Glyphosate and glufosinate concentrations in treatment
solutions used in the terrestrial exposure experiment were
determined by gas chromatography analysis of the TMOAderivatized products using a published procedure (Tseng
et al., 2004). To our knowledge, the method had not been
previously tested on formulated glyphosate or glufosinate
products. This analysis was conducted in order to compare
these measured concentrations to nominal concentrations
(determined gravimetrically based on product label information). Calibration standards, calibration checks, and end
calibration check standards were all constructed using
certified glyphosate and glufosinate stocks obtained commercially (AccuStandard Inc.).
3.
Results
New Mexico spadefoot survival was affected by herbicide
formulation (χ24 = 106.21, P b 0.0001) and substrate (χ21 = 4.95,
P = 0.03), but there was no herbicide formulation–substrate
interaction present (χ24 = 1.79, P = 0.77). After 48-h, New Mexico
Fig. 2 – The survival (mean ± 1 standard error) of juvenile Bufo
cognatus (Great Plains toad) 48-h after direct exposure to aged
well water (control) or an herbicide at the given rate: Roundup
Weed and Grass Killer Ready-To-Use Plus® (WGKP), 1.33 mL
glyphosate/m2; Roundup Weed and Grass Killer Super
Concentrate® (WGKC), 1.33 mL glyphosate/m2; Roundup
WeatherMAX® (WM), 0.16 mL glyphosate/m2; Ignite® 280 SL
(IG), 0.21 mL glufosinate/m2. Animals were exposed in plastic
tubs lined with soil.
Table 3 – Pair-wise comparisons of mean survival of
juvenile Bufo cognatus (Great Plains toad) 48-h after
direct exposure to aged well water (control) or an
herbicide a in plastic tubs lined with paper towel or soil
Paper towel
Contrast
b
Control vs. IG c
Control vs WM
Control vs WGKC
Control vs WGKP
2
Soil
2
df
χ
P
χ
P
1
1
1
1
1.18
0.22
7.04
54.00
0.23
0.64
0.01
b 0.0001
0.01
0.01
0.01
20.21
0.91
0.91
0.91
b0.0001
The degrees of freedom (df), test statistic (χ2) and associated
probability (P) are given.
a
For herbicide application rates see Table 2.
b
Means were compared with a CONTRAST statement in GENMOD.
c
For herbicide formulation codes see Table 1.
spadefoot survival was greater on soil than on paper towel
(Fig. 1). Post-hoc contrasts were used to compare treatment
means (survival) between control animals and those exposed
to each of four herbicide formulations. These analyses
indicated that New Mexico spadefoots exposed to WGKP
exhibited lower survival (Fig. 1, Table 2) than control animals.
All New Mexico spadefoots exposed to WGKP on paper towel
and soil died within 48-h of exposure.
Survival of Great Plains toad was also affected by herbicide
formulation (χ24 = 77.56, P b 0.0001) and substrate (χ21 = 6.99,
P = 0.008). Since a significant herbicide formulation–substrate
interaction was present (χ24 = 14.84, P = 0.0050), data were
separated by substrate and the analysis repeated. These
analyses indicated that survival of Great Plains toads was
affected by herbicide formulation on each substrate (soil:
χ24 = 62.65, P b 0.0001; paper towel: χ24 = 29.75, P b 0.0001). Posthoc contrasts indicated that, compared to control animals,
Great Plains toads exposed to WGKP exhibited greatly reduced
survival on both soil (Fig. 2, Table 3) and paper towel (Fig. 3,
Table 3). Of the Great Plains toad metamorphs exposed to
WGKP on soil, only 22.5% survived for the entire monitoring
period, while all of those exposed on paper towels died within
48 h. Contrasts also indicated that Great Plains toads exposed
to WGKC on paper towel exhibited lower survival compared to
control animals (Fig. 3, Table 3). Only 47.5% of the Great Plains
toads metamorphs exposed to WGKC on paper towel survived
for 48 h. Additional contrasts were used to compare withintreatment survival means between substrates. These analyses
indicated that survival of Great Plains toads was greater
among those exposed to WGKC (Table 4) and WGKP (Table 4)
on soil compared to paper towels.
Analysis of the herbicide solutions used in this study
revealed that, overall, measured concentrations were consistent with nominal values (Table 5) especially considering the
uncertainty associated with the use of the derivatization
method (Tseng et al., 2004) on formulated products. However,
some difficulty was encountered during these tests as the
treatment solutions contained both the active ingredient and
the “inerts”. In some instances, it appeared that these inert
ingredients interfered with the derivatization reaction, particularly for WGKC and IG. Multiple attempts to alter the ratio of
derivatization reagent to sample improved some of the
analyses; however, the IG sample was particularly difficult.
Please cite this article as: Dinehart SK, et al, Toxicity of a glufosinate- and several glyphosate-based herbicides to juvenile
amphibians from the Southern High Plains, USA, Sci Total Environ (2008), doi:10.1016/j.scitotenv.2008.10.010
ARTICLE IN PRESS
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SC IE N CE OF TH E T O T AL E N V I RO N ME N T XX ( 2 0 08 ) XXX–X XX
Table 5 – Accuracy (relative error) of analyses of glyphosate
and glufosinate treatment solutions
Herbicide
formulation
WM b
WGKP
WGKC
IG
a
b
Fig. 3 – The survival (mean ± 1 standard error) of juvenile Bufo
cognatus (Great Plains toad) 48-h after direct exposure to aged
well water (control) or an herbicide at the given rate: Roundup
Weed and Grass Killer Ready-To-Use Plus® (WGKP), 1.33 mL
glyphosate/m2; Roundup Weed and Grass Killer Super
Concentrate® (WGKC), 1.33 mL glyphosate/m2; Roundup
WeatherMAX® (WM), 0.16 mL glyphosate/m2; Ignite® 280 SL
(IG), 0.21 mL glufosinate/m2. Animals were exposed in plastic
tubs lined with paper towel.
In contrast, we experienced no difficulties with the derivatization and subsequent analysis of the individual active
ingredients.
4.
Discussion
Ultimately, we want to understand whether pesticides negatively impact amphibian communities. To achieve this goal,
there must be a transition from highly artificial laboratory
experiments toward research completed under more realistic
conditions (Relyea et al., 2005; Relyea, 2005a). Since we
included natural soil as an exposure substrate, our study
represents an important step in this direction. We exposed
individuals of two recently metamorphosed SHP amphibian
species to environmentally relevant concentrations of a
variety of herbicide formulations while housed on moist
paper towels and natural soil. We used formulated herbicides
Table 4 – Pair-wise comparisons of mean survival of
juvenile Bufo cognatus (Great Plains toad) on soil versus
paper towel 48-h after direct exposure to aged well water
(control) or an herbicide a
Contrast b
df
χ2
P
Control
IG c
WM
WGKC
WGKP
1
1
1
1
1
0.00
1.43
0.33
7.64
12.42
1.00
0.23
0.56
0.0057
0.0004
The degrees of freedom (df), test statistic (χ2) and associated
probability (P) are given.
a
For herbicide application rates see Table 2.
b
Means were compared with a CONTRAST statement in GENMOD.
c
For herbicide formulation codes see Table 1.
Nominal
concentration
(mg/L)
Measured
concentration
(mg/L) a
Relative
error (%)
30
1014
954
41
29.8
1092
628
138
− 0.67
7.7
−34
236
Mean of 3 determinations.
For herbicide formulation codes see Table 1.
because these are the chemicals that juvenile amphibians
encounter in their natural habitats. The survival of both
species tested was reduced only by exposure to those
formulations not intended for agricultural application.
Effects of glyphosate-based herbicide exposure on postmetamorphic amphibians have been examined in just a few
studies, and no data exist for the effects of glufosinate-based
herbicides. Adult and newly metamorphosed Crinia insignifera, a southwestern Australian frog species, exposed to
Roundup 360® exhibited 48-h LC50 values ranging between
65.9 and 69.1 mg glyphosate/L (Bidwell and Gorrie, 1995; Mann
and Bidwell, 1999). Frogs in this study were exposed by partial
submersion to a solution of aged tap water and Roundup 360®.
Relyea (2005a) sprayed juveniles of three North American
amphibian species, while housed on moist paper towels, with
Roundup Weed and Grass Killer® (1.9% glyphosate) at a rate of
1.6 mL glyphosate/m2 to assess the effects of unintended
overspray during agricultural applications. Survival of all
three North American species (Rana sylvatica, Bufo woodhousii
fowleri, and Hyla versicolor) was greatly reduced within 24 h, as
only 32%, 14%, and 18%, respectively, of exposed animals
survived. We exposed SHP playa amphibians to several
glyphosate-based herbicide formulations at a similar or
lower rate (WGKC and WGKP, 1.33 mL glyphosate/m2; WM,
0.16 mL glyphosate/m2) on paper towels and soil. It is
unknown how the composition of WGKP compares to the
formulation used by Relyea (2005a). Our results show that
WGKP reduced survival of both species tested on both
substrates, while WGKC reduced survival of only Great Plains
toads exposed on paper towel. WM had no effect on 48-h
survival of either species tested on either substrate.
Unexpectedly, the response of juveniles amphibians
exposed to WGKC versus WGKP differed. While both formulations were applied at the same rate (1.33 mL glyphosate/m2),
mean survival of both species was dramatically reduced on
both substrates only among animals exposed to WGKP. The
only known difference between the two formulations is that
WGKP contains pelargonic and related fatty acids, suggesting
these compounds are the ingredients responsible for mortality
in our study, not glyphosate or the surfactants included in the
inert ingredients. Pelargonic acid is a natural fatty acid that
acts as an herbicide by quickly desiccating plant tissues (Pline
et al., 2000). Although toxicity testing with pelargonic acid
revealed little or no toxicity toward non-target organisms (e.g.,
fish, birds, honeybees) (U.S. Environmental Protection Agency,
2000), our results indicate that further evaluations of
Please cite this article as: Dinehart SK, et al, Toxicity of a glufosinate- and several glyphosate-based herbicides to juvenile
amphibians from the Southern High Plains, USA, Sci Total Environ (2008), doi:10.1016/j.scitotenv.2008.10.010
ARTICLE IN PRESS
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SC IE N CE O F THE TOTA L E N VI RON ME N T XX ( 2 00 8 ) XXX–X XX
pelargonic acid toxicity in amphibians may be warranted.
Finally, because WGKP and WGKC contain other proprietary
ingredients, we cannot discount the possibility that the
mortality arising from exposure to the formulations could be
explained by the presence of unidentified “inert” ingredient(s)
(e.g., the surfactant).
Because glyphosate, glufosinate, and POEA surfactant bind
rapidly to soil (Giesy et al., 2000; Malone et al., 2004; Lee et al.,
2005) and therefore become less biologically available for
uptake, one would expect that juvenile amphibian survival
would be greater in soil lined compared to paper towel lined
containers. To our knowledge, no previous work has
addressed this question. Relyea (2005a) demonstrated that
the presence of soil in aquatic mesocosms did not mitigate the
toxicity of Roundup Weed and Grass Killer® toward amphibian
larvae, stating that any protective effects of soil were probably
superseded by the rapid onset of tadpole death. Our results
indicate that, in general, New Mexico spadefoots exhibited
greater survival on soil compared to on paper towel. Survival
of Great Plains toads exposed to WGKP or WGKC was also
greater on soil. These results illustrate the importance of
including natural environmental factors when investigating
the effects of pesticides on amphibians (Relyea, 2005a,b).
Failure to do so can lead to inaccurate conclusions about the
risk that these chemicals pose to non-target organisms in field
situations.
The herbicide formulations evaluated in this study vary
widely in their intended use. WGKC and WGKP are commonly
applied to residential lawn and gardens, whereas WM and IG
are commercial agricultural products. WGKP was the only
product that significantly reduced survival among both
species tested for both substrates. Users of this product need
to be aware of the importance of avoiding direct application to
terrestrial amphibians. WGKC also reduced the survival of
Great Plains toads exposed on paper towel. Since WGKP and
WGKC are not intended for agricultural use, results related to
these formulations reveal little about how post-metamorphic
playa amphibians are affected by the application of common
agricultural herbicides. We included these formulations since
previous work (Relyea, 2005a) examining the affects of
glyphosate-based herbicide on terrestrial amphibians used
products intended for lawn and garden use. It seems more
relevant to evaluate the toxicity of agricultural formulations
that amphibians are likely exposed to in field situations
(e.g., WM and IG). These formulations did not reduce survival
at 48-h following exposure for either playa amphibian species
tested. Our results indicate that when the agricultural
formulations examined in this study are used as intended
they do not pose an immediate risk to Great Plains toads or
New Mexico spadefoots.
Although the current study increases our understanding
of how common herbicides impact post-metamorphic
amphibians, it also highlights areas that merit further
research. Impacts of herbicide exposure on survival of
post-metamorphic amphibians were examined in only two
common playa species. Previous research with larval
amphibians has demonstrated that variation in herbicide
sensitivity exists between species (Mann and Bidwell, 1999).
It is therefore prudent to determine how commonly applied
agrochemicals impact juveniles of other amphibian species.
Also, our study only examined the effects of a “worst-case”
exposure level. We chose this single dose because we framed
our study within a tiered approach to ecological risk
assessment (Romeis et al., 2008). Our work represents a
lower tier study used to determine whether the potential for
risk exists. If a lower tier study such as ours indicates the
potential for risk, higher tier studies that more accurately
reflect real-world exposure scenarios should be undertaken
(Romeis et al., 2008). While our results indicate that the
agricultural formulations tested did not pose a threat to
juvenile New Mexico spadefoot and Great Plains toads, many
abiotic and biotic factors present in amphibian habitats were
absent during our study. Previous work has demonstrated
that the toxicity of pesticides toward amphibians changes
when additional natural stressors (e.g., predators) are
present (Relyea, 2003; Relyea et al., 2005). Therefore, further
research completed under increasingly natural conditions is
necessary to understand whether common herbicides pose
any risk toward amphibian populations. Additionally, the
current study monitored only a single endpoint (survival) for
a short period of time. Previous studies have shown that
pesticides can have sublethal impacts on amphibians by
negatively affecting growth (Howe et al., 2004), behavior
(Bridges, 1997) and reproduction (Hayes et al., 2002). The vast
majority of studies examining such sublethal effects have
focused on larval amphibians. More work is needed to
determine whether pesticide exposure causes sublethal
impacts on post-metamorphic amphibians, and what implication such effects have in terms of the persistence of
amphibian populations (Relyea, 2005a).
5.
Conclusion
Many amphibian species occur in areas where pesticide use is
common. While extensive research has examined how these
chemicals impact amphibian larvae, few studies have investigated how pesticide exposure affects post-metamorphic
amphibians. We exposed juveniles of two Southern High
Plains amphibian species to environmentally relevant concentrations of several widely used herbicides. Natural soil was
included as a substrate to increase environmental realism.
Roundup Weed and Grass Killer Ready-to-Use Plus®, an
herbicide intended for lawn and garden use, caused significant mortality among both species. The agricultural formulations (Roundup WeatherMAX® and Ignite® 280 SL) that
juvenile amphibians likely encounter in real-world scenarios
did not affect the short-term survival of either species tested.
While these agricultural herbicides likely do not pose an
immediate threat to the species tested, further research is
needed to determine whether exposure to these herbicides
causes more subtle, sublethal affects.
Acknowledgements
We thank Edward Black, John Allen Jones, and Karlee Martin
for their assistance with these experiments. Funding was
provided by the Caesar Kleberg Foundation for Wildlife
Conservation.
Please cite this article as: Dinehart SK, et al, Toxicity of a glufosinate- and several glyphosate-based herbicides to juvenile
amphibians from the Southern High Plains, USA, Sci Total Environ (2008), doi:10.1016/j.scitotenv.2008.10.010
ARTICLE IN PRESS
SC IE N CE OF TH E T O T AL E N V I RO N ME N T XX ( 2 0 08 ) XXX–X XX
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amphibians from the Southern High Plains, USA, Sci Total Environ (2008), doi:10.1016/j.scitotenv.2008.10.010